This unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells. The first (basic) protocol isolates RNA ...
Preparation of Yeast RNA
UNIT 13.12
This unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells. The first (basic) protocol isolates RNA directly from intact yeast cells by extraction with hot acidic phenol. This procedure yields RNA that is relatively free of contaminating DNA, is convenient to perform with multiple samples, and gives little or no sample-to-sample variation. In contrast, the first alternate protocol relies upon disruption of cells by vigorous mixing with glass beads and denaturing agents. Although this procedure results in efficient breaking of the cells, the product is associated with residual DNA, and the procedure itself is troublesome when one is working with multiple samples. A second alternate protocol describes the scaling up of the first two procedures to isolate enough total RNA for poly (A)+ RNA preparation. NOTE: Take precautions to avoid contamination by RNases. See solutions, for instructions.
UNIT 4.1,
reagents and
PREPARATION OF YEAST RNA BY EXTRACTION WITH HOT ACIDIC PHENOL
BASIC PROTOCOL
Yeast RNA can be isolated efficiently and directly from intact cells by extraction with acidic phenol (pH 5) and SDS at 65°C. Because this procedure does not require vortexing individual samples with glass beads (alternate protocol), which is tedious and a source of variability, it is well-suited for obtaining reproducible quantities of RNA from multiple samples. In addition, RNA preparations are largely devoid of contaminating DNA which partitions into the interface during the extraction step. Materials Yeast cells and desired medium (UNITS 13.1 & 13.2) TES solution Acid phenol Chloroform 3 M sodium acetate, pH 5.3 100% and 70% ethanol, ice-cold 50-ml centrifuge tube (Falcon) Centrifuge: tabletop or Sorvall equipped with an SS-34 rotor Additional reagents and equipment for ethanol precipitation (UNIT 2.1) and spectrophotometric quantitation of cells and RNA (APPENDIX 3) 1. Grow yeast cells in 10 ml of desired medium to mid-exponential phase (OD600 = 1.0). It is not advisable to prepare RNA from cells that have reached a higher density because as the stationary phase is approached, the results are less consistent and RNA yields will vary.
2. Transfer culture to 50-ml centrifuge tube and centrifuge cells 3 min at 1500 × g (7000 rpm in a tabletop centrifuge or SS-34 rotor), 4°C. The time and speed of the centrifugation are not critical.
3. Discard supernatant, resuspend pellet in 1 ml ice-cold water. Transfer to a clean 1.5-ml microcentrifuge tube. Microcentrifuge 10 sec at 4°C, and remove supernatant. Proceed to step 4 or if desired immediately freeze pellet by placing tube in dry ice. Liquid nitrogen may also be used to freeze the pellets. Although not essential, freezing is particularly useful when RNA is to be prepared from multiple cultures or multiple time points from a given culture; this permits simultaneous processing of the samples. The frozen Contributed by Martine A. Collart and Salvatore Oliviero Current Protocols in Molecular Biology (1993) 13.12.1-13.12.5 Copyright © 2000 by John Wiley & Sons, Inc.
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cell pellets can be stored for months at −70°C. Thaw on ice just before continuing the procedure.
4. Resuspend cell pellet in 400 µl TES solution. Add 400 µl acid phenol and vortex vigorously 10 sec. Incubate 30 to 60 min at 65°C with occasional, brief vortexing. It is crucial to incubate for ≥30 min (with occasional vortexing) to obtain quantitative recovery of both large and small RNA species.
5. Place on ice 5 min. Microcentrifuge 5 min at top speed, 4°C. 6. Transfer aqueous (top) phase to a clean 1.5-ml microcentrifuge tube, add 400 µl acid phenol, and vortex vigorously. Repeat step 5. 7. Transfer aqueous phase to a clean 1.5-ml microcentrifuge tube and add 400 µl chloroform. Vortex vigorously and microcentrifuge 5 min at top speed, 4°C. 8. Transfer aqueous phase to a new tube, add 40 µl of 3 M sodium acetate, pH 5.3, and 1 ml of ice-cold 100% ethanol and precipitate. Microcentrifuge 5 min at top speed, 4°C. Wash RNA pellet by vortexing briefly in ice-cold 70% ethanol. Microcentrifuge as before to pellet RNA. 9. Resuspend pellet in 50 µl H2O. Determine the concentration spectrophotometrically by measuring the A260 and A280 (UNIT 4.1). Store at −70°C, or at −20°C if it is to be used within 1 year. Make sure that the RNA is well dissolved; if necessary, heat the resuspended pellet at 65°C for 10 to 20 min and/or dilute further with more water. The yield from 10 ml of cells grown in YPD medium is ∼300 ìg. Cells grown in less optimal medium will yield less RNA per ml culture. ALTERNATE PROTOCOL
PREPARATION OF RNA USING GLASS BEADS Yeast RNA is also efficiently released by disrupting the cells using high-speed mixing in the presence of glass beads and denaturing agents. Proteins are removed by extraction with organic solvents and the RNA is recovered by ethanol precipitation and quantitated by measuring its absorbance at 260 nm. This preparation is suitable for S1, northern hybridization, or primer extension analyses (UNITS 4.6, 4.9, and 4.8, respectively) and can be prepared quickly and easily from a relatively small quantity of yeast cells. Although the RNA isolated by this procedure is contaminated with DNA, the DNA component does not interfere with most analytical studies. Additional Materials RNA buffer 25:24:1 phenol/chloroform/isoamyl alcohol (equilibrated with RNA buffer; see support protocol, UNIT 2.1) 0.45- to 0.55-mm, chilled, acid-washed glass beads (Sigma) Prepare the cells 1. Grow and process yeast cells and freeze cell pellet as in steps 1 to 3 of the basic protocol. If necessary, the samples can now be quick-frozen on dry ice and stored at −70°C. Thaw on ice just before processing.
2. Resuspend pellet in 300 µl RNA buffer. Preparation of Yeast RNA
Disrupt the cells 3. Add a volume of chilled acid-washed glass beads equivalent to ∼200 µl water.
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Prepare acid-washed glass beads by soaking in concentrated nitric acid for 1 hr, washing extensively with deionized water, and drying in a baking oven. Before use the glass beads should be chilled on ice. Manipulate the beads with a stainless steel spatula that has been dried in a baking oven. Do this carefully, using a spatula or a funnel fashioned out of weighing paper. If beads stick to the lip of the microcentrifuge tube, they will prevent the tube from closing securely.
4. Add 300 µl of 25:24:1 phenol/chloroform/isoamyl alcohol equilibrated with RNA buffer. A phenol mix previously equilibrated with TE buffer (UNIT 2.1) can be reequilibrated with RNA buffer. Remove the aqueous layer (TE buffer), add a volume of RNA buffer equal to the volume of the organic layer, shake vigorously, and allow the phases to separate. Remove the aqueous layer (RNA buffer), add fresh RNA buffer, mix well, and allow the phases to separate.
5. Close the cap, then invert and shake up and down to ensure that the beads are suspended. Vortex vigorously for 2 min at highest speed. Hold 2 to 4 tubes on the head of a vortexer for 1 min, place these tubes on ice, and vortex another set. After 1 min with the second set, place on ice, and vortex the first set for another minute. This limits the processing to 8 samples at one time. If a high-speed horizontal shaker is available many samples can be vortexed simultaneously. Use the highest speed setting for 3 min in a cold room.
6. Microcentrifuge 1 min at room temperature. Transfer aqueous (top) layer to a clean microcentrifuge tube. Avoid the interface by taking only the uppermost 200 to 250 ìl from each sample. Taking a fixed amount from each sample results in a more uniform yield of RNA from each sample.
7. Add an equal volume of 25:24:1 phenol/chloroform/isoamyl alcohol (200 to 250 µl). Vortex vigorously 10 sec. 8. Repeat step 6. Precipitate the RNA 9. Add 3 vol (∼600 µl) of ice-cold 100% ethanol. Mix well and place at −20°C for ≥30 min or on dry ice for 5 min. 10. Microcentrifuge 2 min at 4°C. Aspirate or pour off the supernatant and wash pellet with ice-cold 70% ethanol. 11. Microcentrifuge 1 min at 4°C. Aspirate or pour off supernatant and dry pellet. 12. Resuspend pellet in 50 µl H2O. Determine the concentration spectrophotometrically by measuring the A260 and A280 (UNIT 4.1). Store the RNA at −70°C. If 2 × 108 cells are used, the RNA concentration of the final solution will be ∼2 mg/ml.
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ALTERNATE PROTOCOL
PREPARATION OF POLY(A)+ RNA The RNA isolated in the basic and first alternate protocols is also a suitable source of poly(A)+ RNA for use in constructing cDNA libraries. For this purpose, larger quantities of RNA can be isolated by simply scaling-up the procedures. For 1010 S. cerevisiae cells, use the following guidelines: For the hot acidic phenol protocol: Increase volumes of TES and acid phenol solutions to 4 ml each; use 50-ml polypropylene centrifuge tubes for all manipulations. The yield will be ∼10 mg of RNA. For the glass beads protocol: Increase volumes to 15 ml RNA buffer, 10-ml volume glass beads, 15 ml phenol/chloroform/isoamyl alcohol, and 30 ml of 100% ethanol. Perform the procedure in a 50-ml disposable polypropylene tube and centrifuge 10 min, 3000 rpm (1200 × g), 4°C in a fixed-angle or swinging-bucket type rotor at each phenol extraction step. Good recovery of the precipitated nucleic acid can be accomplished by centrifugation under these same conditions. The yield will be ∼5 mg total RNA. Prepare poly(A)+ RNA from either method using the protocol presented in UNIT 4.5. REAGENTS AND SOLUTIONS Acid phenol Add sufficient water to a bottle of solid phenol such that phenol is water-saturated; pH will be ∼5.0. Do not buffer phenol. Store at 4°C, protected from light. RNA buffer 0.5 M NaCl 200 mM Tris⋅Cl, pH 7.5 10 mM EDTA Store indefinitely at room temperature TES solution 10 mM Tris⋅Cl, pH 7.5 10 mM EDTA 0.5% SDS Store indefinitely at room temperature COMMENTARY Background Information
Preparation of Yeast RNA
Aside from the harsh conditions used to break open yeast cells, the RNA isolation procedures presented here are similar to the phenol/SDS method for isolating RNA from plant cells (UNIT 4.3). Both methods take advantage of the fact that phenol extraction is an effective means of inactivating and removing RNases. The hot acidic phenol method is preferable when working with multiple samples; because the number of manipulations and time required for each are reduced, very little sample-to-sample variation is encountered. The glass bead disruption procedure causes the release of both RNA and DNA, and the absorbance at 260 nm will measure total nucleic acid. For precise determination of RNA con-
centrations, the DNA component can be removed as described in UNITS 4.1, 4.3, and 4.5. This is not necessary for routine northern blot, primer extension, and S1 analyses. Refer to UNITS 4.1-4.5 for further information on the purification and properties of RNA.
Critical Parameters As with all RNA manipulations, precautions must be taken to prevent RNase contamination. See UNIT 4.1 for details. In the hot acidic phenol method, the two phenol and one chloroform extractions are critical to obtain clean RNA for analysis. These protocols are designed to process multiple samples in tandem—for example, when monitoring gene induction over time or
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when examining RNA synthesis driven by different promoter constructions. In these cases (where quantitation is critical), detection using probes in northern hybridization, S1 mapping, or primer extension studies should be carried out in parallel (preferably in the same tube or hybridization bag) with a probe for a gene in which transcription does not change over time or in response to inducing conditions. The ratios of signal intensities for the two probes in each sample are then compared.
Anticipated Results The yield of RNA isolated using the hot acidic phenol protocol is ∼300 µg from 2 × 108. The yield resulting from the glass bead disruption protocol starting with 2 × 108 cells will be ∼100 µg of total RNA. Scale-up of either method will produce ∼5 to 10 mg total RNA. Ten to twenty micrograms of total RNA isolated by either procedure is sufficient for most northern blot, S1 mapping, or primer extension applications.
Time Considerations Depending on the organization of the researcher, 12 to 24 samples can be processed conveniently in about 1 hr using the hot acidic phenol method. For the glass bead method, eight samples can be easily processed without using a high-speed shaking apparatus in ∼1.5 hr. A second set of eight samples can be processed while the first set is precipitating at − 20°C. With a high-speed shaking apparatus 16 samples can be processed in about 1 hr. Both phenol extraction steps can be done on the shaking apparatus. If RNA synthesis is being monitored over time, samples from individual time points can be frozen at ∼70°C (see step 3, basic protocol) so that all of the samples can be processed simultaneously.
Contributed by Martine A. Collart and Salvatore Oliviero Harvard Medical School Boston, Massachusetts
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